IS 5960-12: Meat and Meat Products - Methods of Test, Part 12: Determination of L-(+)-Glutamic Acid Content - Reference Method

17 Pages·2001·1.3 MB·English

by  Bureau of Indian Standards

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timely dissemination of this information in an accurate manner to the public.
“जान1 का अ+धकार, जी1 का अ+धकार” “प0रा1 को छोड न’ 5 तरफ”
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“The Right to Information, The Right to Live” “Step Out From the Old to the New”
IS 5960-12 (2001): Meat and Meat Products - Methods of
Test, Part 12: Determination of L-(+)-Glutamic Acid Content
- Reference Method [FAD 18: Slaughter House and Meat
Industry]
“!ान $ एक न’ भारत का +नम-ण”
Satyanarayan Gangaram Pitroda
““IInnvveenntt aa NNeeww IInnddiiaa UUssiinngg KKnnoowwlleeddggee””
“!ान एक ऐसा खजाना > जो कभी च0राया नहB जा सकता हहहहै””ै”
Bhartṛhari—Nītiśatakam
“Knowledge is such a treasure which cannot be stolen”

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Indian Standard
MEAT AND MEAT PRODUCTS —
METHODS OF TEST
PART 12 DETERMINATION OF L-(+)-GLUTAMIC ACID CONTENT — REFERENCE METHOD
( First Revision)
ICS 67.120.10
..

0 BIS 2001
BUREAU OF INDIAN STANDARDS
MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI 110002

August 2001 Price Group 6
_
Slaughter House and Meat industry Sectional Committee, FAD 56 --3
NATIONAL FOREWORD
This Indian Standard (Part 12) (First Revision) which isidentical withISO4134: 1999 ‘Meat and meat
products — Determination of L-(+) -glutamic acid content -– Reference method’, issued by the
International Organization for Standardization (ISO) was adopted by the Bureau of Indian Standards
on the recommendation of Slaughter House and Meat Industry Sectional Committee and approval of
the Food and Agriculture Division Council.
This standard was originally published in 1988 based on the earlier version of the International
Standard, namely, ISO 4134:1978 under dual numbering. This is being revised to align with the
revised version, namely, ISO 4134:1999.
In the adopted standard, certain terminology and conventions are not identical to those used in the
Indian Standards. Attention is particularly drawn to the following:
a) Wherever the words ‘International Standard’ appear referring to this standard, they should be
read as ‘Indian Standard’; and
b) Comma (,) has been used as adecimal marker while in Indian Standards, the current practice
is to use a point (.) as the decimal marker.
CROSS REFERENCES
Inthis adopted standard, the following International Standards are referred to. Read intheir respective
place, the following:
international Standard Corresponding Indian Standard Degree of
Equivalence
ISO 648 : 1977 Laboratory IS 1117 : 1975 One-mark pipettes (first Related
glassware — One-mark revision)
pipettes

ISO 835-2 : 1981 Laboratory IS 4162 (Part 2) :1985 Graduated pipettes: Equivalent
glassware — Graduated Part 2 Pipettes for which no waiting time is
pipettes — Part 2: Pipettes for specified (first revision)
which no waiting time is
>
specified
ISO 1042 : 1998 Laboratory IS 915: 1975 One-mark volumetric flasks Related
glassware — One-mark (first revision)
volumetric flasks
ISO 1442:1996 Meat and meat IS 5960 (Part 5) :2001 Meat and meat Identical
products — Determination of products — Methods of test: Part 5
moisture content (Reference Determination of moisture content
method)
ISO 3100-1 : 1991 Meat and ls/lso 3100-1 : 1991 Meat and meat do
meat products — Sampling and products — Method of sampling
preparation of test samples —
Part 1: Sampling
For the purpose of deciding whether a particular requirement of this standard is complied with the
final value, observed or calculated, expressing the result of atest or analysis, shall be rounded off in
accordance with IS 2 : 1960 ‘Rules for rounding of numerical values (reviseO)’. The number of
significant places retained inthe rounded off value, should be the same as that of the specified value
inthis standard.
IS 5960 [ Part 12 ) :2001
1s0 4134:1999
Indian Standard
,...’,
MEAT AND MEAT PRODUCTS —
METHODS OF TEST 1
PART 12 DETERMINATION OF L-(+)-GLUTAMIC ACID CONTENT — REFERENCE METHOD
(First Revision )
1 Scope
This International Standard specifies a reference method for the determination of the L-(+)-glutamic acid content of
meatand meatproducts, including poultry.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged to
investigate the possibility of applying the most recent editions of the normative documents indicated below. For
undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC
maintain registers of currently valid International Standards.
ISO 648, Laboratory glassware — One-mark pipettes.
ISO 835-2, Laboratory glassware — Graduated pipettes — Part 2: Pipettes for which no waiting time is specified.
ISO 1042, Laboratory glassware — One-mark volumetric f/asks.

ISO 1442, Meat and meat products — Determination of moisture content (Reference method).

3 Terms and definitions
For the purposes of this International Standard, the following.term and definition apply.
3.1
L-(+)-glutamic acid content of meat and meat products
mass fraction of L-(+)-glutamic acid determined according to the procedure described inthis International Standard
NOTE The L-(+)-glutamic acid content isexpressed inpercent.
4 Principle
The L-(+)-ghJtamic acid present in a test portion is extracted with perchloric acid solution at a temperature of O“C.
The ‘extract is centrifuged, decanted and filtered and the pH is adjusted to 10,0. Nicotinamide adenine dinucleotide
(NAD) is reduced by the L-(+)-glutamic acid in the presence of glutamate dehydrogenase [equation (1)]. The
resultant reduced nicotinamide adenine dinucleotide (NADH) is reacted with iodonitrotetrazolium chloride in the
presence of diaphorase [equation (2)]. The resulting formazane is measured at a wavelength of 492 nm and the
L-(+)-ghtamic acid content iscalculated.
IS 5960 ( Part 12 ) :2001
1s0
4134 ; 1999
glutamate dehydrogenase
L-(+)-glutamate + NAD+ + H20 a-ketoglutamate + NADH + NH3 + H+ (1)
,
diaphorase
NADH + iodonitrotetrazolium chloride + H+ ~ NAD+ + formazane (2)
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified
Except for the solutions of inorganic compounds (5.2 and 5.3), store all solutions in stoppered brown glass bottles
which have been scrupulously cleaned and steamed or sterilized.
5.1 Water, double-distilled, or demineralized and distilled water, obtained by carrying out the final distillation in an
all-glass apparatus.
NOTE Water distilled onlyonce may contain metal iontraces, and demineralized water maycontain microorganisms. Metal
ions may decrease the activity ofenzymes, while microorganisms may give rise to an aspecific enzymatic background activity
that might adversely affect the results ofanalysis.
5.2 Dilute perchloric acid, c(HC104) = 1,0 moi/1.
WARNING — Contact with oxidizable or combustible materials or with dehydrating or reducing agents may
result in fire or explosion. Persons using this acid should be thoroughly familiar with its hazards. See
safety practices listed in annex A.
Dilute 8,6 ml of perchloric acid, 70 O/.(by mass), p20= 1,67 g/ml, to 100 ml with water (5.1). Add the perchloric acid
to the bulk of the water before the final dilution to volume and mix.
5.3 Potassium hydroxide solution, c(KOH) = 2 mol/1,
Dissolve 56,1 g of potassium hydroxide in water (5.1). Dilute when cooled to 500 ml and mix.

5.4 Triethanolamine phosphate buffe r solution, pH = 8,6.

Dissolve 1,86 g of triethanolamine hydrochloride in water (5.1) and adjust the pH to 8,6 with the potassium
hydroxide solution (5.3) using a pH-meter. Add 0,68 g of octylphenol decaethyleneglycol ether (e.g. Triton X-100).
Dilute to 100 ml with water and mix (solution A).
Dissolve 0,86 g of dipotassium hydrogen phosphate (K2HP04) and 7 mg of potassium dihydrogen phosphate
(KH2P04) in water (5.1). Dilute to 100 ml with water and mix (solution B).
Mix 20 ml of solution Awith 5 ml of solution B.
The solution is stable for 2 months when stored at atemperature of between O‘C and 6 “C.
5.5 Nicotinamide adenine dinucleotide (NAD) sofution.
Weigh 25 mg of NAD in a small, stoppered flask. Add 5,0 ml of water (5.1) and mix.
The solution is stable for 2 months when stored in the dark at atemperature of between O“C and 6 ‘C.
5.6 Iodonitrotetrazolium chloride (lNT) solution, 2-(4-iodophenyl) -3-(4-nitrophenyl) -5-phenyltetrazolium chloride.
Weigh 30 mg of INT in a small, stoppered brown flask. Add 50 ml of water (5.1) and mix.
The solution is stable for 4weeks when stored inthe dark at atemperature of between O“C and 6 “C

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IS 5960 ( Part 12 ) :2001
1s0 4134:1999
5.7 Diaphorase solution (Iipoamide dehydrogenase, ECIJ 1.8.1.4).
Dissolve 3 mg of Iyophilized diaphorase in 1ml of water (5.1) and mix.
The solution isstable for 4weeks when stored at a temperature of between O“C and 6 ‘C.
5.8 Glutamate dehydrogenase (GLDH) solution (EC1J1.4.1.3), 10 mg/ml, free from ammonium sulfate,
ethylene-dinitrilotetraacetic acid (EDTA) and glutaminase.
This solution is supplied as such (for example in quantities of 1,0 ml) and is stable for 12 months when stored at a
temperature of between O‘C and 6 ‘C.
5.9 L-(+)-glutamic acid, standard solution.
Dissolve 50,0 mg of L-(+)-glutamic acid (C5H904N) in 25 ml of water (5.1). Adjust the pH to 7,0 with potassium
hydroxide solution (5.3). Dilute to 50 ml and mix.
Keep this solution at atemperature of between O‘C and 6 ‘C and dilute 1+ 49 shortly before use.
6 Apparatus
Usual laboratory equipment and, in particular, the following.
6.1 Mechanical or electrical equipment, capable of homogenizing the laboratory sample.
This includes a high-speed rotational cutter, or a mincer fitted with a plate with apertures not exceeding 4,0 mm in
diameter.
6.2 Laboratory mixer.
6.3 Laboratory centrifuge, with 50 ml or 100 ml centrifuge tubes, operating at a radial acceleration of about
2000 g“.
6.4 pH-meter.
6.5 Fluted filter papers, of diameter about 15 cm, high or moderate speed.

6.6 One-mark volumetric flasks, of capacities 100 ml and 250 ml, complying with ISO 1042, class B.

6.7 One-mark pipettes, of capacities 100 ml, 50 ml and 25 ml, complying with ISO 648, class B.
6.8 Gra5uated (automatic) pipettes, for delivering 2.50 ml, 0,50 ml, 0,20 ml and 0,05 ml, complying with
ISO 835-2, class A.
6.9 Small plastics spatula, bent at 90°, for mixing the contents of the cuvettes.
6.10 Photoelectric calorimeter, provided with a filter having a transmittance maximum at a wavelength of
492 nm, or spectrometer.
6.11 Cuvettes, of 10 mm optical path length.
6.12 Analytical balance, capable of weighing to the nearest 0,1 mg.
‘J The EC number refers to the Enzyme Classification number as given in:The International Union of Biochemistry, ,%zyrne
norrrenc/ature,Elsevier, Amsterdam, 1965.

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IS 5960 ( Part 12 ) :2001 i
1s0 4134:1999
7 Sampling
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Sampling is not part of the method specified in this International Standard. A recommended sampling method is
given in ISO 3100-1.
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
Start from a representative sample of at least 200 g. Store the sample in such a way that deterioration and change
in composition are prevented.
8 Preparation of test sample
Homogenize the laboratory sample with the appropriate equipment (6.1). Take care that the temperature of the
sample material does not rise above 25 ‘C. If a mincer is used, pass the sample at least twice through the
equipment.
Fill a suitable airtight container with the prepared sample. Close the container and store in such a way that
deterioration and change in composition are prevented. Analyse the sample as soon as practicable, but always
within 24 h after homogenization.
9 Procedure
NOTE If it is required to check whether the repeatability requirement is met, carry out two single determinations in
accordance with 9.1 to9.3.
9.1 Test portion
Weigh, to the nearest 0,01 g, approximately 50 g of the test sample (see clause 8) and transfer this test portion to
the jar of the laboratory mixer (6.2).
9.2 Preparation of extract
9.2.1 Add 100 ml of dilute perchloric acid (5.2) at a temperature of O“C to the test portion and homogenize the

mixture.

9.2.2 Transfer a portion of the homogenate to a centrifuge tube (6.3). Centrifuge for 10 min at a radial acceleration
of about 2000 gn. Carefully move aside the fat layer and decant the supernatant liquid through a fluted filter paper
(6.5) into a 200 ml conical flask. Discard the first 10 ml of the filtrate.
9.2.3 Transfer by pipette (6.7) 50 ml of the solution (which should be only slightly turbid) into a 100 ml beaker and
adjust the pH to 10,0 with the potassium hydroxide solution (5.3) using the pH-meter (6.4).
9.2.4 Transfer the contents of the beaker quantitatively into a 100 ml volumetric flask (6.6). Dilute to the mark with
water (5.1)and mix.
9.2.5 Cool the solution in ice for 10 rein, and filter through a fluted filter paper (6.5). Discard the first 10 ml of the
filtrate.
9.2.6 Pjpette 25 ml, or some other appropriate volume (V), of the filtrate into a 250 ml volumetric flask (6.6) and
dilute to the mark with water.
NOTE The volume Vshould be chosen sothatthe L-(+)-glutamic acid content ofthe solution isless than 30 mg/1,
IS 5960 ( Part 12 ) :2001
1s0 4134:1999 —...+
..-1
$ 9.3 Determination
9.3.1 Bring the buffer solution (5.4) and the filtrate (9.2.6) to a temperature of 20 ‘C to 25 “C.
) Pipette into each of two cuvettes (6.11) 2,50 ml of the buffer solution (5.4), 0,20 ml of the NAD solution (5.5),
0,20 ml of the INT solution (5.6) and 0,05 ml of the diaphorase solution (5.7).
After the addition of INT, restrict exposure of the reaction mixture to light to an unavoidable minimum.
Into one of the cuvettes, pipette 0,50 ml of the filtrate (9.2.6); the solution obtained isthe test solution.
Into the other cuvette, pipette 0,50 ml of water (5.1); the solution obtained isthe blank solution.
Mix the solutions with the spatula (6.9) and read the absorbance AI of each cuvette at a wavelength of 492 nm
against water. The temperature of the solution shall be 20 ‘C to 25 ‘C.
9.3,2 Pipette 0,05 ml of the GLDH solution (5.8) into each of the cuvettes. Mix the contents of the cuvettes by
swirling or by moving the spatula up and down.
Read the absorbance A2 of each cuvette at a wavelength of 492 nm against water after 10 min to 15 min and every
2 min thereafter until a constant increase in absorbance is obtained. Plot the absorbance against time. Extrapolate
the absorbance values to the moment of start of the reaction (see annex B).
9.3.3 Repeat the operations described in 9.3.1 and 9.3.2, but replace the 0,50 ml of filtrate (9.2.6) in the first
cuvette by 0,50 ml of the L-(+)-glutamic acid standard solution (5.9).
9.3.4 Determine the moisture content of the test sample according to ISO 1442.
10 Calculation and expression of results
10.1 Absorbance difference for standard solution
Calculate the absorbance difference for the standard solution using the following equation:

A&= (A2~– Al~)–(A2b-Alb)

where
MS isthe absorbance difference for the standard solution;
Alb isthe absorbance of the blank solution, measured in 9.3.3 in accordance with 9.3.1;
A2b isthe absorbance of the blank solution, measured in 9.3.3 in accordance with 9.3.2;
A1~ isthe absorbance of the standard solution, measured in 9.3.3 in accordance with 9.3.1;
A2~ isthe absorbance of the standard solution, measured in 9.3.3 in accordance with 9.3.2.
10.2 Absorbance difference for test solution
Calculate the absorbance difference for the test solution using the following equation:
AA=(A2-A, )-(A2~-A, ~)
where
AA isthe absorbance difference for the test solution;

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